Abstract
The interaction of ethidium, a DNA intercalator, with the poly(dA).poly(dT) duplex and the poly (dA).2poly(dT) triplex has been investigated by a variety of spectrophotometric and hydrodynamic techniques. The fluorescence of ethidium is increased when either the duplex or triplex form is present. Binding constants, determined from absorbance measurements, indicate that binding to the triple helical form is substantially stronger than to the duplex, with a larger binding site size (2.8 base triplets compared to 2.4 base pairs). Furthermore, while binding to poly(dA).poly(dT) shows strong positive cooperativity, binding to the triplex is noncooperative. Thermal denaturation experiments demonstrate that ethidium stabilizes the triple helix. Binding to either form induces a weak circular dichroism band in the visible wavelength region, while in the region around 310 nm, there is a band that is strongly dependent on the degree of saturation of the duplex, and which is positive for the duplex but negative for the triplex. Both fluorescence energy transfer and quenching studies provide evidence of intercalation of ethidium in both duplex and triplex complexes. Binding of ethidium leads to an initial decrease in viscosity for both the duplex and triplex structures, followed by an increase, which is greater for the duplex. Taken together, these results strongly suggest that ethidium binds to the poly (dA).2poly(dT) triple helix via an intercalative mechanism.
Highlights
With the poly(dA)*poly(dT) duplex and the poly These studieshave important practical implicationins diverse*Bpoly(dT)triplex has been investigated by a variety of spectrophotometric and hydrodynamic techniques
Fi[5,6,7] on the H-foromf DNA observed in supercoiledplasmids, nally, there have been repor(t3s)that ethidiumdoes not bind explained in termsof an intramolecular triplexp, rovided the totriple helices such as poly(dCdT.)poly(dAdG.)poly first evidence of a biologically significant role for DNA triple, which are stabilized at low pH, where the third helices
In the experiments described below, we present evidence, basedon viscosity, circular dichroism, fluorescence quenching, andenergy transfer data, which suggests that ethidium binds to ptohley(dA) .2poly(dT) triple helix by an intercalative mechanism
Summary
With the poly(dA)*poly(dT) duplex and the poly These studieshave important practical implicationins diverse (dA)*Bpoly(dT)triplex has been investigated by a variety of spectrophotometric and hydrodynamic techniques. Binding of ethidium leads to an initial decrease in viscosity for both the duplex and triplex structures, followed by an increase, which is greater for the duplex. Taken together, these results strongly suggest that ethidium binds to the poly (dA)*2poly(dT)triple helix via an intercalative mechanism. Le Pecqand coworkers [27, 28] studiedthe interaction of ethidium with the same triplexmore quantitatively, determining the binding constant and stoichiometry They observed that the affinity of ethidium for the triplex form was lower than that for the duplex poly(rA).poly(rU), which is consistent with the repofrta decreasein the melting temperature for the third strandof the triplex. Helene [17,18] have focused on the development of artificial bound ligand increases the stability of the oligopyrimidine
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